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United States Patent |
5,523,391
|
Komurasaki
,   et al.
|
June 4, 1996
|
DNA fragment encoding tumor cell growth inhibitors
Abstract
DNA fragments encoding a tumor cell growth inhibitors and having a
nucleotide sequence shown by formula (1) below, which are produced by
preparing cDNA library from mRNA of the established 3T3 cell-derived cell
line, amplifying various DNA fragments considered to encode the tumor cell
growth inhibitors by the PCR method, analyzing the nucleotide sequences of
these DNA fragments and determining the nucleotide sequences of the DNA
fragments encoding the inhibitors:
##STR1##
wherein X represents TTC TTT CTA or TTC (SEQ ID NO:1 or SEQ ID NO:2).
Inventors:
|
Komurasaki; Toshi (Tokyo, JP);
Toyoda; Hitoshi (Tokyo, JP);
Yoshimoto; Makoto (Tokyo, JP);
Hanada; Kazunori (Tokyo, JP)
|
Assignee:
|
Taisho Pharmaceutical Co., Ltd. (JP)
|
Appl. No.:
|
244309 |
Filed:
|
May 25, 1994 |
PCT Filed:
|
December 3, 1992
|
PCT NO:
|
PCT/JP92/01580
|
371 Date:
|
May 25, 1994
|
102(e) Date:
|
May 25, 1994
|
PCT PUB.NO.:
|
WO93/11233 |
PCT PUB. Date:
|
June 10, 1993 |
Current U.S. Class: |
536/23.5; 435/69.1; 435/252.3; 435/320.1; 530/324; 536/23.1 |
Intern'l Class: |
C12N 015/28; C12N 015/19; C12N 015/12 |
Field of Search: |
536/23.1,23.5
530/324
435/69.1,172.3,320.1,252.3
|
References Cited
U.S. Patent Documents
4675285 | Jun., 1987 | Clark et al. | 435/6.
|
5281520 | Jan., 1994 | O'Hara et al. | 435/69.
|
5384394 | Jan., 1995 | Komurasaki et al. | 530/324.
|
Other References
C. C. Lee et al. Science 239:1288-1291 (Mar. 1988).
J. M. Wozney Meth. in Enzymol. 182:738-751 (1990).
J. Cell Physiol., vol. 119, 1984, pp. 101-106 Harel et al.
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Prouty; Rebecca
Attorney, Agent or Firm: Lorusso & Loud
Claims
We claim:
1. A DNA fragment encoding a tumor cell growth inhibitor which has a
nucleotide sequence shown by formula (1):
##STR4##
wherein X represents TTC TTT CTA or TTC (SEQ ID NO:1 or SEQ ID NO:2).
Description
TECHNICAL FIELD
The present invention relates to DNA fragments encoding novel tumor cell
growth inhibitors. More particularly, the present invention relates to DNA
fragments encoding novel tumor cell growth factors which can be obtained
from the culture supernatant of 3T3 cell-derived cell line and exhibit an
activity of inhibiting the growth of tumor cells.
BACKGROUND ART
Synthetic drugs such as chemotherapeutic agents or immunotherapeutic agents
have been heretofore widely used as anti-tumor agents. However, these
drugs generally encounter problems that their specificity is low and
side-effects are serious. On the other hand, many tumor cell growth
inhibitors have been found in tissue culture cells. These inhibitors could
be such anti-tumor agents that would be highly specific and would have
minimized side-effects. Representative examples of such inhibitors are
interferon, lymphotoxin and tumor necrosis factor (TNF). Recently, a tumor
cell cytotoxic factor obtained from human fibroblast and a tumor cell
growth inhibitor obtained from human lung cancer cells are reported in
Japanese Patent KOKAI Nos. 1-148197 and 1-187094, respectively.
Some cell growth inhibitors are isolated also from the fibroblastic 3T3
cell line established from the cells obtained from Swiss fetal mice. For
example, Natraj et al. has reported that a growth inhibitor was obtained
from the cell surface of 3T3 cells in the stationary phase, cf., Proc.
Natl. Acad. Sci. U.S.A., 75, 6115-6119 (1978). Harel et al. has reported
that a growth inhibitor having a molecular weight of 40 kDa was obtained
from the culture supernatant of 3T3 cells, see J. Cell. Physiol., 119,
101-106 (1984), ibid., 123, 139-143 (1985). However, these growth
inhibitors all fail to show any significant inhibitory action against
tumor cells, as is known in the art.
The present inventors previously succeeded in isolating, from the culture
supernatant of 3T3 cell-derived cell line, novel tumor cell growth
inhibitors having an activity of inhibiting the growth of tumor cells,
which was filed as Japanese Patent Application No. 3-11950.
The tumor cell growth inhibitors exhibit a potent growth inhibition
activity against human promyelogenous leukemia cells or human uterine
cervical cancer-derived cells and are expected to be effective for the
treatment of cancer.
For use as new carcinostatic agents, it is required to supply the tumor
cell growth inhibitors in a sufficient amount. It is thus desired to
develop a method for production available with industrial advantages.
DISCLOSURE OF INVENTION
The present inventors have brought attention to recombinant DNA technique
applicable to the process for production of the tumor cell growth
inhibitors in an industrially efficient way, and made investigations on
cloning of cDNA encoding the tumor cell growth inhibitors. Succeeded by
recombinant DNA technique in obtaining DNA fragments encoding the
inhibitors which can be used for production of the inhibitors, the present
invention has been accomplished.
That is, the present invention relates to DNA fragments encoding the tumor
cell growth inhibitors, which has nucleotide sequence shown by formula
(1):
##STR2##
wherein X represents TTC TTT CTA or TTC.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing elution profile of phenyl 5PW-RP reversed phase
HPLC of tumor cell growth inhibitor P-1, which is the subject of the
present invention.
FIG. 2 is a graph showing elution profile of phenyl 5PW-RP reversed phase
HPLC of tumor cell growth inhibitor P-2 which is the subject of the
present invention.
FIG. 3 shows oligonucleotides (1) through (11) used as primers in the PCR
method.
FIG. 4 briefly shows the amplified site of DNA fragment amplified by the
PCR method.
FIG. 5 is an outline of the DNA fragment obtained by the PCR method.
FIG. 6 shows a nucleotide sequence of the DNA fragment encoding a part of
P-1and the translated amino acid sequence.
FIG. 7 shows a nucleotide sequence of the DNA fragment encoding the
C-terminal region of P-1 and the translated amino acid sequence.
FIG. 8 shows a nucleotide sequence of the DNA fragment encoding the
N-terminal region of P-1 and the amino acid sequence translated therefrom.
FIG. 9 is a photograph showing the results of electrophoresis performed for
separating the DNA fragment encoding the entire region of P-1.
FIG. 10 shows a nucleotide sequence of the DNA fragment encoding the entire
region of P-1 and the amino acid sequence translated therefrom.
BEST MODE FOR CARRYING OUT THE INVENTION
The two tumor cell growth inhibitors are involved in the present invention,
one inhibitor being encoded by the DNA fragment having a nucleotide
sequence of formula (1) wherein X is TTC TTT CTA (hereinafter sometimes
abbreviated as P-1), another being encoded by the DNA fragment having a
nucleotide sequence of formula (1) wherein X is TTC (hereinafter sometimes
abbreviated as P-2).
These inhibitors can be isolated and purified from the culture supernatant
of the established cell line NIH3T3-sf, which is obtained by subculture
from NIH3T3 cells (J. Virol., 4, 549 (1969)), one of fibroblastic 3T3 cell
lines established from Swiss fetal mice, see Japanese Patent Application
No. 3-11950.
The inhibitors P-1 and P-2 are peptides having an amino acid sequence shown
by formula (2) below:
##STR3##
wherein Y represents Phe-Phe-Phe-Leu or Phe.
The inhibitor P-1 is a peptide having an amino acid sequence shown by
formula (2) wherein Y is Phe-Phe-Leu, and the inhibitor P-2 is a peptide
having an amino acid sequence shown by formula (2) wherein Y is Phe.
Cloning of cDNA encoding the inhibitor P-1 or P-2 is performed as described
below.
From NIH3T3-sf cells which are the established cell line, mRNA is
extracted, adsorbed onto oligo dT cellulose column and then eluted to
purify the adsorbed mRNA. These procedures may be carried out using a
commercially available kit for mRNA extraction.
Using the thus purified mRNA as a template and oligo dT as a primer, cDNA
is synthesized by reverse transcriptase and DNA polymerase. The terminus
of this cDNA is rendered blunt in a conventional manner and bound to,
e.g., EcoRI adapter. The product is then blended with, e.g., lambda phage
gt10-EcoRI arm to bind to each other, using T4 DNA ligase. A vector
containing cDNA is thus constructed. Next, phage particles are formed by
the in vitro packaging method (Hohn et al., Proc. Natl. Acad. Sci. U.S.A.,
74, 3259 (1977)) using the vector-bound cDNA as a template to obtain cDNA
library.
Using this cDNA library as a template, various DNA fragments considered to
encode the inhibitor P-1 or P-2 are amplified by the PCR method (Saiki et
al., Science, 230, 1350 (1985)). Based on the thus obtained DNA fragments,
the nucleotide sequence of the desired DNA fragment encoding the inhibitor
P-1 or P-2 can be determined.
More specifically, the cloning can be made following the procedures shown
in the embodiments described below.
Firstly, a DNA fragment which is considered to encode the inhibitor P-1 is
amplified by the PCR method, using as the 5'-end primer the
oligonucleotide (1) shown in FIG. 3 and having the nucleotide sequence
corresponding to 1 to 6 residues of the amino acid sequence and as the
3'-end primer the oligonucleotide (2) shown in FIG. 3 and having the
complementary sequence to the nucleotide sequence which corresponds to 41
to 46 amino acid sequence, in the amino acid sequence of the inhibitor P-1
shown in FIG. 2. In this case, the cDNA library described above is used
as a template. Next, a DNA fragment which is considered to encode a part
of the inhibitor P-1 is further amplified by the PCR method, using as a
template the amplified DNA fragment, which is named the reaction product 1
and, using the oligonucleotide (1) as the 5'-end primer and as the 3'-end
primer the oligonucleotide (3) shown in FIG. 3 and having the
complementary sequence to the nucleotide sequence corresponding to 32 to
37 amino acid residues.
The amplified DNA fragment, which is named the reaction product 2, is
separated by electrophoresis. The thus obtained DNA fragment is cloned to,
e.g., single stranded phage M13mp18RF (Messing et al., Gene, 33, 103
(1985)) and the nucleotide sequence of the DNA fragment is determined by
the dideoxy chain terminator method (Sanger, E. et al., Proc. Natl. Acad.
Sci. U.S.A., 74, 5463 (1977)). FIG. 6 shows the nucleotide sequence of the
DNA fragment thus determined, namely, the DNA fragment encoding the amino
acid sequence of 1 to 36 residues in the inhibitor P-1.
Based on the thus determined nucleotide sequence, a DNA fragment which is
considered to encode the C-terminal region of the inhibitor P-1 is further
amplified by the PCR method. That is, the DNA fragment is amplified in a
manner similar to the procedure described above, using as the 5'-end
primer, e.g., the oligonucleotide (4) (see FIG. 3) corresponding to 18 to
37 bases and the oligonucleotide (5) (see FIG. 3) corresponding to 68 to
87 bases, and as the 3'-end primer the oligonucleotide (6) (see FIG. 3)
having the complementary nucleotide sequence to the sequence near the
EcoRI digestion site of .lambda.gt10 used for producing the cDNA library,
in the nucleotide sequence shown in FIG. 6; in this case, the cDNA library
is used as a template. Based on the thus obtained DNA fragment, which is
named the reaction product 4, the nucleotide sequence is determined as
described above. FIG. 7 shows the nucleotide sequence of the DNA fragment
containing the C-terminal region of the inhibitor P-1.
Next, the nucleotide sequence of the DNA fragment encoding the N-terminal
region of the inhibitor P-1 is determined in a similar manner. That is,
the DNA fragment is amplified by the PCR method using the oligonucleotides
(7), (8) and (9) shown in FIG. 3 as primers. Using the so obtained DNA
fragment, which is named the reaction product 5, the nucleotide sequence
is determined in a similar manner. FIG. 8 shows the nucleotide sequence of
the DNA fragment containing the N-terminal region of the inhibitor P-1.
Based on the nucleotide sequences of various DNA fragments thus obtained
and the amino acid sequence of the inhibitor P-1, the nucleotide sequence
of the DNA fragment encoding the entire region of the inhibitor P-1 can be
determined, see FIG. 10.
Based on the nucleotide sequence so determined, the DNA fragment encoding
the inhibitor P-1 is cloned to obtain the DNA fragment in large
quantities.
That is, the DNA fragment is amplified by the PCR method, using as the
5'-end primer the oligonucleotide (10) shown in FIG. 3 and corresponding
to the 5'-end region of the nucleotide sequence shown in FIG. 10 or by
formula (1) and as the 3'-end primer the oligonucleotide (11) shown in
FIG. 3 and having the complementary sequence to the nucleotide sequence
corresponding to the 3'-end region; in this case, the cDNA library
prepared as described above is used as a template. The amplified DNA
fragment, which is named the reaction product 6, is separated by
electrophoresis. The thus obtained DNA fragment is then cloned to, e.g.,
single stranded phage M13mp18RF at the SmaI site thereof, as described
above. The nucleotide sequence of the DNA fragment of formula (1) which
encodes the inhibitor P-1 can thus be obtained.
The sites at which various DNA fragments are amplified as described above
by the PCR method using the oligonucleotides (1) to (11) as the primers
are illustratively shown in FIG. 4. The various DNA fragments so
amplified, namely, the DNA fragments of the reaction products 2, 4, 5 and
6 described above, are illustratively shown in FIG. 5.
The DNA fragment encoding the inhibitor P-2 can be obtained in a similar
manner. For example, the DNA fragment is amplified by the PCR method,
using as the 5'end primer the oligonucleotide (10) corresponding to the
5'-end region of the nucleotide sequence shown in FIG. 10 and as the
3'-end primer the oligonucleotide (11) having the complementary sequence
(5'-GAAGTGCTCACATCGCAGAC-3') to the nucleotide sequence of 113 to 132
bases at the 3'-end shown in FIG. 10; in this case, the cDNA library
described above is used as a template. The amplified DNA fragment is then
separated by electrophoresis as described above. The thus obtained DNA
fragment is cloned to obtain the DNA fragment encoding the inhibitor P-2.
The cloning of cDNA encoding the inhibitor P-1 and the inhibitor P-2 may
also be effected as follows.
The oligonucleotide deduced from the amino acid sequence shown by formula
(2) is chemically synthesized and labelled with an isotope. Using the
labelled oligonucleotide as a probe, the desired cDNA is isolated, e.g.,
from the cDNA library described above, by the plaque hybridization
technique, and then cloned in a conventional manner.
Alternatively, the DNA fragment having the nucleotide sequence of formula
(1) which encodes the inhibitor P-1 or P-2 may also be chemically
synthesized by known methods, e.g., by the triester phosphate method
(Letsinger et al., J. Am. Chem. Soc., 91, 3350 (1969)).
Hereinafter the present invention will be described below in more detail,
by referring to the examples and the reference examples.
Reference Example Isolation and purification of inhibitors P-1 and P-2 as
well as the determination of their structures
1. Preparation of 3T3 cell-derived established cell line
NIH3T3 cells were subcultured in DF medium (Dulbecco's modified
MEM:HamF-12=1:1) containing 10% calf fetal serum and then cultured in DF
containing 5 .mu.g/ml of insulin, 5 .mu.g/ml of transferrin and
2.times.10.sup.-8 M selenate to obtain proliferated clones.
From the clones, a clone which grew only in DF medium was selected and
subcultured to establish the cell line. The thus obtained cell line was
named NIH3T3-sf. The incubation was performed at 37.degree. C. under the
gaseous phase of 5% CO.sub.2. The subculture was carried out by diluting
to 2-fold at the time when the culture cells reached sub-confluence. The
medium was prepared from a conditioned medium and a fresh medium in a
proportion of 50%:50% and the so prepared medium was provided for use.
2. Preparation of serum-free culture supernatant of NIH3T3-sf cells
NIH3T3-sf cells were cultured in DF medium containing 10% calf fetal serum.
When the cultured cells reached confluence, the medium was removed and
washed once with PBS(-) (KCl:0.2 g, KH.sub.2 PO.sub.4 :0.2 g, NaCl:8 g,
Na.sub.2 HPO.sub.4 : 1.150 g/l) followed by incubation in DF medium for 48
hours. After the medium was removed, incubation was performed in a fresh
DF medium for 96 to 120 hours. The medium was exchanged with fresh medium
every 96 to 120 hours to collect 100 liters. The collected medium was
centrifuged at 2000 r.p.m. for 10 minutes to recover the supernatant.
3. Purification
1) Q-Sepharose column chromatography:
Using Perikon cassette system (ultrafiltering membrane system, molecular
weight for fractionation: 1000), 100 liters of the culture supernatant
collected was concentrated to about 50 times. The concentrate was
subjected to salting-out with 90% ammonium sulfate saturation followed by
centrifugation at 8000.times.g for 60 minutes. The thus obtained
precipitates were dissolved in 20 mM Tris-HCl buffer (pH 7.4) and the
solution was dialyzed the same buffer. Next, the dialysate was added to
Q-Sepharose column (Pharmacia, .phi.5 cm.times.5 cm), which had been
previously equilibrated with the same buffer, to collect the non-adsorbed
fraction and the fraction washed.
Conditions for the elution are as follows.
Flow rate: 8 ml/min
Fractionation: 2 ml/tube
Eluant: 20 mM Tris-HCl buffer (pH 7.4)
2) S-Sepharose column chromatography:
After adjusting pH to 5.0 with acetic acid, the non-adsorbed fraction was
added to S-Sepharose column (Pharmacia, .phi.2.5 cm.times.6 cm), which had
been previously equilibrated with 20 mM acetate buffer (pH 5.0). The
active component was adsorbed onto the column. Elution with 20 mM Tris-HCl
buffer (pH 7.4) to obtain the active fraction. Conditions for the elution
were as follows.
Flow rate: 0.85 ml/min
Fractionation: 4 ml/tube
Eluant: 20 mM Tris-HCl buffer (pH 7.4)
3) Hydroxyapatite column chromatography HPLC:
After adjusting pH of the active fraction eluted out of the S-Sepharose
column to 6.0 with acetic acid, the active fraction was added to
hydroxyapatite column (Asahi Optical Co., Ltd., .phi.7.5 mm.times.10 cm),
which had been previously equilibrated with 20 mM acetate buffer (pH 6.0).
The non-adsorbed fraction was thus collected. Conditions for the elution
were as follows.
Flow rate: 1 ml/min
Fractionation: 1 ml/tube
Eluant: 20 mM acetate buffer (pH 6.0)
4) TSK gel CM-3SW column chromatography HPLC:
After adjusting pH of the active fraction to 5.0 with acetic acid, the
fraction was poured onto TSK gel CM-3SW column (Toso, .phi.7.5
mm.times.7.5 cm), which had been previously equilibrated with 20 mM
acetate buffer (pH 5.0) containing 5% acetonitrile (CH.sub.3 CN).
Conditions for the elution were as follows.
Flow rate: 1 ml/min
Fractionation: 1 ml/tube
Eluant:
(A) 20 mM acetate buffer (pH 5.0)/5% CH.sub.3 CN
(B) 20 mM acetate buffer (pH 5.0)/5% CH.sub.3 CN/0.2M NaCl linear density
gradient of A.fwdarw.B (120 minutes)
The activity was noted in the two fractions which were eluted in NaCl
concentrations of 86 mM (P-1) and 100 mM (P-2).
5) Phenyl 5PW-RP reversed phase column chromatography HPLC:
The active fractions obtained in the CM-3SW HPLC step were poured onto
Phenyl-5PWRP column (Toso, .phi.4.6 mm.times.7.5 cm), respectively, which
had been previously equilibrated with 20 mM acetate buffer (pH 7.4)
containing CH.sub.3 CN. Elution was effected by eluting with 20% CH.sub.3
CN-containing 5 mM phosphate buffer (pH 7.4) for 20 minutes and then by
linear density gradient for 80 minutes using the same buffer containing
20% to 40% CH.sub.3 CN. The flow rate was 1 ml/min and fractionation was
performed at 2 ml/tube. P-1 and P-2 were eluted at the positions of 59 to
60 minutes, and 60 to 61 minutes in retention time, respectively, see
FIGS. 1 and 2.
5. Determination of amino acid sequences
The amino acid sequences of the two products purified were determined by
the automated Edman degradation method using a gaseous protein sequencer
(Model 470A, Applied Bio-Systems Co., Ltd.). As described above, the
determination revealed that P-1 has an amino acid sequence of formula (2)
wherein Y is Phe-Phe-Leu and P-2 has an amino acid sequence of formula (2)
wherein Y is Phe.
EXAMPLE
Isolation of DNA fragments encoding P-1 and P-2 and determination of the
nucleotide sequences
1) Production of cDNA library of NIH3T3-sf cells
(1) Preparation of NIH3T3-sf cells:
NIH3T3-sf cells were cultured in the manner shown in Reference Example 2.
That is, the cells were cultured at 37.degree. C. in 10% calf fetal
serum-containing DF medium in 5% CO.sub.2. When the cells reached
confluence, the medium was removed and washed once with PBS (-) followed
by incubation in DF medium for 120 hours.
(2) Extraction of mRNA from NIH3T3-sf cells:
The medium of the cells cultured in the manner shown in (1) above was
removed. After washing once with PBS (-), PBS (-) was supplemented. The
cells were then scraped out with a cell scraper and collected in a conical
tube. After centrifugation at 1500.times.g for 5 minutes at room
temperature, PBS (-) was added to suspend the cells therein. The
suspension was again centrifuged to obtain the precipitates. From the
precipitates, mRNA was extracted using mRNA Extraction Kit (manufactured
by Invitrogen Co., Ltd.). Following this procedure, 19.2 .mu.g of mRNA was
purified from 2.times.10.sup.8 cells.
(3) Synthesis of cDNA:
Using the mRNA prepared in (2) as a template, cDNA was synthesized using
oligo dT as a primer, by the use of cDNA Synthesis Kit (manufactured by
Pharmacia). Following this procedure, 1.0 .mu.g of cDNA was synthesized
from 1.9 .mu.g of mRNA.
(4) Binding of cDNA to vector:
cDNA Cloning Kit (manufactured by Pharmacia) was used. That is, after the
terminus of the cDNA synthesized in (3) above was rendered blunt with DNA
polymerase large fragment of E. coli and four deoxynucleotide
triphosphates, EcoRI adapter was bound thereto.
The cDNA was mixed with lambda phage gt10-EcoRI arm (manufactured by
Strategene Co., Ltd.) and bound to each other using T4 DNA ligase.
(5) In vitro packaging:
Using the vector-bound cDNA shown in (4) as a template, phage particles
were produced using in vitro packaging kit (manufactured by Amersham Co.,
Ltd.) to prepare cDNA library.
2) Amplification of DNA fragment encoding a part of P-1 by the PCR method
and analysis of nucleotide sequence
(1) Amplification of DNA fragment encoding a part of P-1:
Based on the amino acid sequence of P-1 determined in Reference Example: 5,
oligonucleotides corresponding to the amino acid sequences of the
N-terminal and C-terminal regions were synthesized. Using these
oligonucleotides as primers, the DNA fragment encoding a part of P-1 was
amplified by the PCR method (Saiki et al., Science, 230, 1350 (1985)), in
which the cDNA library prepared in 1) above was used as a template. In the
PCR reaction, Gene Amp PCR reagent Kit with AmpliTaq DNA Polymerase
(manufactured by Perkin-Elmer Cetus Instrument Co., Ltd.) and DNA Thermal
Cycler (manufactured by Perkin-Elmer Cetus Instrument Co., Ltd.) were
used.
That is, oligonucleotides having the following nucleotide sequences in the
amino acid sequence of P-1 shown in formula (2) were synthesized, see
FIGS. 3 and 4:
oligonucleotide (1) having the nucleotide sequence corresponding to the
amino acid sequence of 1 to 6 residues (Val-Gln-Ile-Thr-Lys-Cys):
5'-GTNCARATHACNAARTG-3'
wherein N is A, T, G or C; R is A or G; H is A, C or T: a mixture of the
oligonucleotides wherein N, R and H represent the respective bases was
used;
oligonucleotide (2) having the complementary sequence to the nucleotide
sequence corresponding to the amino acid sequence of 41 to 46 residues
(Cys-Glu-His-Phe-Phe-Leu):
5'-ARRAARAARTGYTCRCA-3'
wherein Y is C or T; a mixture of the two oligonucleotides wherein Y is C
or T was used; oligonucleotide (3) having the complementary sequence to
the nucleotide sequence corresponding to the amino acid sequence of 32 to
37 residues (Cys-Glu-Val-Gly-Thy-Thr):
5'-GTRTANCCNACYTCRCA-3'
Next, the DNA fragment encoding a part of P-1 was amplified by the
following procedures.
______________________________________
heating 10 .mu.l of the cDNA library prepared in 1)-
(5) above at 100.degree. C. for 10 minutes
.dwnarw.
ice cooling
.dwnarw.
adding thereto:
oligonucleotide (1) in a final
concentration of 10 .mu.M
oligonucleotide (2) in a final
concentration of 10 .mu.M
0.5 .mu.l (2.5 units) of AmpliTaq Polymerase
(manufactured by Perkin-Elmer Cetus
Instrument Co., Ltd. ) and,
distilled water to make the whole volume
100 .mu.l
.dwnarw.
adding thereto:
10 .mu.l of 10 .times. Buffer A (100 mM Tris-HC1, pH
8.3, 500 mM KC1, 15 mM MgCl.sub.2, 0.01% (w/v)
gelatin) and,
16 .mu.l of 1.25 mM dNTP (wherein N is A, T, G
or C)
.dwnarw.
heating at 94.degree. C. for 1 minute
.dwnarw.
heating at 40.degree. C. for 2 minutes
.dwnarw.
heating at 72.degree. C. for 3 minutes
(30 repetitions of the heating procedure)
.dwnarw.
reaction product 1
10 .mu.l of the reaction product 1
.dwnarw.
adding thereto:
oligonucleotide (1) in a final
concentration of 10 .mu.M
oligonucleotide (3) in a final
concentration of 10 .mu.M
0.5 .mu.l (2.5 units) of AmpliTaq Polymerase
(manufactured by Perkin-Elmer Cetus
Instrument Co., Ltd.)
and distilled water to make the whole
volume 100 .mu.l
.dwnarw.
adding thereto:
10 .mu.l of 10 .times. Buffer A and
16 .mu.l of 1. 25 mM dNTP (wherein N is A, T, G
or C)
.dwnarw.
heating at 94.degree. C. for 1 minute
.dwnarw.
heating at 40.degree. C. f or 2 minutes
.dwnarw.
heating at 72.degree. C. for 3 minutes
(30 repetitions of the heating procedure)
.dwnarw.
reaction product 2 (cf. FIG. 5)
______________________________________
(2) Cloning of DNA fragment encoding a part of P-1:
The reaction product 2 was subjected to electrophoresis on 5%
polyacrylamide gel, whereby a band stained with ethydium bromide was
confirmed around 110 base pairs. The band was cut out and cloned to single
stranded phage M13mp18RF at the SmaI site. The cutting-out of the band and
extraction of DNA were carried out as follows, according to T. Maniatis et
al., Molecular Cloning, page 178 (1982).
______________________________________
dissolving DNA in 7 .mu.l of H.sub.2 O
.dwnarw.
adding to the solution:
1 .mu.l of 10 .times. Buffer B (0.5M Tris-HC1, pH
7.8, 0.1 M MgCl.sub.2, 10 mM DTT)
1 .mu.l (5 units) of Klenow fragment and,
1 .mu.l of 10 mM dNTP wherein N is A, T, G or C
.dwnarw.
heating at 22.degree. C. for 1 hour
.dwnarw.
heating at 68.degree. C. for 10 minutes
.dwnarw.
ethanol precipitation
.dwnarw.
dissolving the precipitates in 7 .mu.l of distilled
water
.dwnarw.
adding to the solution:
1 .mu.1 of 10 .times. Buffer C (0.5M Tris-HC1, pH
7.6, 0.1M MgCl.sub.2, 0.1M DTT)
1 .mu.l (5 units) of 10 mM ATP and,
1 .mu.l of T4 polynucleotide kinase
.dwnarw.
heating at 37.degree. C. for 1 hour
.dwnarw.
heating at 68.degree. C. for 10 minutes
.dwnarw.
phenol extraction
.dwnarw.
ethanol precipitation
.dwnarw.
dissolving the precipitates in 7 .mu.l of distilled
water
.dwnarw.
adding to the solution:
1 .mu.l of 10 .times. Buffer D (0.66M Tris-HC1, pH
7.6, 50 mM MgCl.sub.2 50 mM DTT, 10 mM ATP)
1 .mu.l (350 units) of T4 DNA ligase and,
1 .mu.l (0.5 .mu.g) of M13mp18RF digested with
SmaI
.dwnarw.
heating at 15.degree. C. for 15 hours
______________________________________
This DNA was transfected to E. coli JM109 (C. Yanisch-Perrson et al., Gene.
33, 103 (1985)) treated with calcium chloride (Maniatis et al., Molecular
Cloning, 250 (1982)) and then seeded on L agar medium (trypton: 10 g,
yeast extract: 5 g, sodium chloride: 10 g, agar powders: 15 g/l). To 3 ml
of soft agar medium (trypton: 10 g, yeast extract: 5 g, sodium chloride:
10 g, agarose: 7.5 g/l) kept at 45.degree. C., was added 0.1 ml of E. coli
JM109 independently incubated. The mixture was laid on the L agar medium
plate and incubated at 37.degree. C. to obtain a plaque.
(3) Analysis of nucleotide sequence:
The plaque prepared in 2)-(2) was adsorbed onto strain JM109 followed by
incubation. From the culture supernatant single stranded DNA was extracted
according to the method of Messing et al., Gene, 33, 103 (1985). Using
7-Deaza-Sequencing Kit (Toyobo Co., Ltd.), the nucleotide sequence was
determined by the dideoxy chain terminator method (Sanger, F. et al.,
Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977)), cf. FIG. 6. Translation
of the nucleotide sequence into amino acids reveals that the DNA fragment
encodes a part (1 to 36 amino acid residues) of P1.
3) Amplification of DNA fragment encoding the C-terminal region of P-1 by
the PCR method and analysis of its nucleotide sequence
(1) Amplification of the DNA fragment encoding the C-terminal region of
P-1:
Based on the nucleotide sequence determined in 2)-(3), oligonucleotides
were synthesized. Using these oligonucleotides and a part of .lambda.gt10
as primers, the DNA fragment encoding the C-terminal region of P-1 was
amplified by the PCR method in which the cDNA library prepared in 1)-(5)
above was used as a template. That is, the following oligonucleotides were
synthesized, see FIGS. 3 and 4.
oligonucleotide (4) corresponding to 18 to 37 bases, in the nucleotide
sequence shown in FIG. 4;
oligonucleotide (5) corresponding to 68 to 87 bases, and
oligonucleotide (6) having the nucleotide sequence around the EcoRI
digestion site of .lambda.gt10.
Next, the DNA fragment encoding the C-terminal region of P-1 was amplified
by the procedure shown below.
______________________________________
heating 10 .mu.l of the CDNA library prepared in 1)-
(5) above at 100.degree. C. for 10 minutes
.dwnarw.
ice cooling
.dwnarw.
adding thereto:
oligonucleotide (4) in a final
concentration of 1 .mu.M
oligonucleotide (6) in a final
concentration of 1 .mu.M
0.5 .mu.l (2.5 units) of AmpliTaq Polymerase
(manufactured by Perkin-Elmer Cetus
Instrument Co., Ltd.) and,
distilled water to make the whole volume
100 .mu.l
.dwnarw.
adding thereto 10 .times. Buffer A and 16 .mu.l of 1.25 mM
dNTP (wherein N is A, T, G or C)
.dwnarw.
heating at 94.degree. C. for 1 minute
.dwnarw.
heating at 52.degree. C. for 2 minutes
.dwnarw.
heating at 72.degree. C for 3 minutes
(30 repetitions of the heating procedure)
.dwnarw.
reaction product 3
10 .mu.l of the reaction product 3
.dwnarw.
adding thereto:
oligonucleotide (5) in a final
concentration of 1 .mu.M
oligonucleotide (6) in a final
concentration of 1 .mu. M
0.5 .mu.l (2.5 units) of AmpliTaq Polymerase
(manufactured by Perkin-Elmer Cetus
Instrument Co., Ltd. )
and distilled water to make the whole
volume 100 .mu.l
.dwnarw.
adding thereto 10 .times. Buffer A and 16 .mu.l of 1.25 mM
dNTP (wherein N is A, T, G or C)
.dwnarw.
heating at 94.degree. C. for 1 minute
.dwnarw.
heating at 55.degree. C. for 2 minutes
.dwnarw.
heating at 72.degree. C. for 3 minutes
(30 repetitions of the heating procedure)
.dwnarw.
reaction product 4 (cf. FIG. 5)
______________________________________
(2) Cloning of DNA fragment encoding the C-terminal region of P-1:
The reaction product 4 was subjected to electrophoresis on 5%
polyacrylamide gel, whereby a band stained with ethydium bromide was
confirmed around 400 base pairs. The band was cut out and cloned to single
stranded phage M13mp18RF at the SmaI site by the procedure shown in
2)-(2).
(3) Analysis of nucleotide sequence:
The nucleotide sequence was determined by the procedure shown in 2)-(3)
above, see FIG. 7. Translation of the nucleotide sequence into amino acids
reveals that the DNA fragment encodes the C-terminal region of P-1. That
is, the DNA fragment encodes the C-terminal region corresponding to the
underlined 24 to 46 amino acid residues in FIG. 7.
4) Amplification of DNA fragment encoding the N-terminal region of P-1 by
the PCR method and analysis of its nucleotide sequence
(1) Amplification of the DNA fragment encoding the N-terminal region of
P-1:
Based on the nucleotide sequence of P-1 determined in 2)-(3),
oligonucleotides were synthesized. Using these oligonucleotides and a part
of .lambda.gt10 as primers, the DNA fragment encoding the N-terminal
region of P-1 was amplified by the PCR method in which the cDNA library
prepared in 1)-(5) above was used as a template.
That is, the following oligonucleotides were synthesized, see FIGS. 3 and
4.
oligonucleotide (7) having the complementary sequence to the nucleotide
sequence corresponding to 18 to 37 bases, in the nucleotide sequence of a
part of P-1 shown in FIG. 4;
oligonucleotide (8) having the complementary sequence to the nucleotide
sequence corresponding to 68 to 87 bases, and
oligonucleotide (9) having the nucleotide sequence around the EcoRI
digestion site of .lambda.gt10.
Next, the DNA fragment encoding the N-terminal region of P-1 was amplified
in a manner similar to the procedure shown in Example 2)-(1) except that
the oligonucleotides (8) and (7) were used instead of the oligonucleotides
(4) and (5), and the oligonucleotide (9) was used instead of the
oligonucleotide (6). Finally the reaction product 5 was obtained, see FIG.
5.
(2) Cloning of DNA fragment encoding the N-terminal region of P-1:
The reaction product 5 was subjected to electrophoresis on 5%
polyacrylamide gel, whereby a band stained with ethydium bromide was
confirmed around 310 base pairs. The band was cut out and cloned to single
stranded phage M13mp18RF at the SmaI site by the procedure shown in
2)-(2).
(3) Analysis of nucleotide sequence:
The nucleotide sequence was determined by the procedure shown in 2)-(3)
above, see FIG. 8. Translation of the nucleotide sequence into amino acids
reveals that the DNA fragment encodes the N-terminal region of P-1. That
is, the DNA fragment encodes the N-terminal region corresponding to the
underlined 1 to 12 amino acid residues in FIG. 8.
5) Verification of the nucleotide sequence of the DNA fragment encoding the
entire region of P-1 by the PCR method
In order to confirm the nucleotide sequence of the DNA fragment encoding
the entire region of P-1 in the nucleotide sequences determined in 2)
through 4) above, the oligonucleotide (10) corresponding to the 5'-end
region and the oligonucleotide (11) having the complementary sequence to
the nucleotide sequence corresponding to the 3'-end were synthesized, see
FIGS. 3 and 4. Using these oligonucleotides as primers, the amplification
and cloning of the DNA fragment encoding the entire region of P-1 were
performed to analyze its nucleotide sequence.
(1) Amplification of the DNA fragment encoding the entire region of P-1:
______________________________________
heating 10 .mu.l of the cDNA library prepared in 1)-
(5) above at 100.degree. C. for 10 minutes
.dwnarw.
ice cooling
.dwnarw.
adding thereto:
oligonucleotide (10) in a final
concentration of 1 .mu.M
oligonucleotide (11) in a final
concentration of 1 .mu.M
0.5 .mu.l (2.5 units) of AmpliTaq Polymerase
(manufactured by Perkin-Elmer Cetus
Instrument Co., Ltd.)
and distilled water to make the whole
volume 100 .mu.l
.dwnarw.
adding thereto 10 .times. Buffer A and 16 .mu.l of 1.25 mM
dNTP (wherein N is A, T, G or C)
.dwnarw.
heating at 94.degree. C. for 1 minute
.dwnarw.
heating at 55.degree. C. for 2 minutes
.dwnarw.
heating at 72.degree. C. for 3 minutes
(30 repetitions of the heating procedure)
.dwnarw.
reaction product 6 (see FIG. 5)
______________________________________
(2) Cloning of DNA fragment encoding the entire region of P-1:
The reaction product 6 was subjected to electrophoresis on 5%
polyacrylamide gel, whereby a band stained with ethydium bromide was
confirmed around 138 base pairs, see FIG. 9. The band was cut out and
cloned to single stranded phage M13mp18RF at the SmaI site by the
procedure shown in 2)-(2).
(3) Analysis of nucleotide sequence:
The nucleotide sequence was determined by the procedure shown in 2)-(3)
above, see FIG. 10. Translation of the nucleotide sequence into amino
acids reveals that the amino acid sequence of the DNA fragment fully
coincided with the entire amino acid sequence of P-1.
P-2 has such a structure that the 2 amino acid residues are deleted from
the C-terminus of P-1. Accordingly, cloning of the DNA fragment encoding
the entire region of P-2 can be performed by the procedures similar to
those in 5) (1) and (2), using as primers oligonucleotide (10)
corresponding to the 5'-end used in 5)-(1) and oligonucleotide
(5'-GAAGTGCTCACATCGCAGAC-3') having the complementary sequence to the
nucleotide sequence corresponding to the 3'-end of the nucleotide sequence
encoding P-2.
Industrial Applicability
As described above in detail, the present invention can provide the DNA
fragments encoding the novel tumor cell growth inhibitors which are
expected to be effective for the treatment of leukemia and uterus cervical
cancer. By transfecting the DNA fragments to an expression vector in,
e.g., E. coli and culturing the transformant obtained, the tumor cell
growth inhibitors can be produced in large quantities. Therefore, the DNA
fragments of the present invention enable to produce the tumor cell growth
inhibitors in an industrial scale.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 16
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTGCAGATTACAAAGTGTAGTTCTGACATGGACGGCTACTGCTTGCATGGCCAGTGCATC60
TACCTGGTGGACATGAGAGAGAAATTCTGCAGATGTGAAGTGGGCTACACTGGTCTGCGA120
TGTGAGCACTTCTTTCTA 138
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GTGCAGATTACAAAGTGTAGTTCTGACATGGA CGGCTACTGCTTGCATGGCCAGTGCATC60
TACCTGGTGGACATGAGAGAGAAATTCTGCAGATGTGAAGTGGGCTACACTGGTCTGCGA120
TGTGAGCACTTC132
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ValGlnIleThrLysCysSerSerAspMetAspGlyTyrCysLeuHis
151015
GlyGlnCysIleTyrLeuValAspMetArgGluLysPheCysArgCys
202530
GluValGlyTyrThrGlyLeuArgCysGluHisPhePheLeu
354045
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ValGlnIleThrLysCysSerSerAspMetAspGlyTyrCysLeuHis
151015
Gl yGlnCysIleTyrLeuValAspMetArgGluLysPheCysArgCys
202530
GluValGlyTyrThrGlyLeuArgCysGluHisPhe
35 40
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TAGTTCTGACATGGACGGCT 20
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TGGACATGAGAGAGAAATTC 20
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGAGTATTTCTTCCAGGG 19
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AGCCGTCCATGTCA GAACTA20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GAATT TCTCTCTCATGTCCA20
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AGCAAGTTCAGCCTGGTTAA20
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GTGCAGATTACAAAGTGTAG20
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TAGAAAGAAGTGCTCACATC20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TGGACATGAGAGAGAAATTCTGCAGATGTGAAGTGGGCTACACTGGTCTGCGA53
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AspMetArgGluLysPheCysArgCysGluValGlyTyrThrGlyLeu
151015
Arg
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GCGACTTGGTGGGCTCTGGTACCTGGATCAGCTCGGTTCCAACTCAGCCACAGG5 4
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
AlaThrTrpTrpAlaLeuValProGlySerAlaArgPheGln LeuSer
151015
HisArg
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